Comparative genomic analyses of four novel Ramlibacter species and the cellulose-degrading properties of Ramlibacter cellulosilyticus sp. nov.

In this study, four novel bacterial strains, USB13T, AW1T, GTP1T, and HM2T, were isolated from various environments in Busan and Jeju Island, Republic of Korea. The 16S rRNA sequencing results indicated that the four novel strains belong to the genus Ramlibacter. All four strains were tested for their potential cellulolytic properties, where strain USB13T was identified as the only novel bacterium and the first within its genus to show cellulolytic activity. When tested, the highest activities of endoglucanase, exoglucanase, β-glucosidase, and filter paper cellulase (FPCase) were 1.91 IU/mL, 1.77 IU/mL, 0.76 IU/mL, and 1.12 IU/mL, respectively at pH 6.0. Comparisons of draft whole genome sequences (WGS) were also made using average nucleotide identity, digital DNA-DNA hybridization values, and average amino acid identity values, while whole genome comparison was visualized using the BLAST Ring Image Generator. The G + C contents of the strains ranged from 67.9 to 69.9%, while genome sizes ranged from 4.31 to 6.15 Mbp. Based on polyphasic evidence, the novel strains represent four new species within the genus Ramlibacter, for which the names Ramlibacter cellulosilyticus sp. nov. (type strain, USB13T = KACC 21656T = NBRC 114839T) Ramlibacter aurantiacus sp. nov. (type strain, AW1T = KACC 21544T = NBRC 114862T), Ramlibacter albus sp. nov. (type strain, GTP1T = KACC 21702T = NBRC 114488T), and Ramlibacter pallidus sp. nov. (type strain, HM2T = KCTC 82557T = NBRC 114489T) are proposed.

Phylogenetic and genomic analyses. The 16S rRNA genes of strains USB13 T , AW1 T , GTP1 T , and HM2 T were sequenced through Sanger sequencing using the universal bacterial primer sets 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) as well as 518F (5′-CCA GCA GCC GCG GTA ATA C-3′) and 805R (5′-GAC TAC CAG GGT ATC TAA TC-3′), while the sequences were obtained through Sanger sequencing, as previously described by Kim et al. 32 . The nearly complete 16S rRNA genes were assembled using SeqMan software (DNASTAR Inc., Madison, WI, USA), and their similarities to each other, and also to closely related type strains were calculated using National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) searches 33 and the EzBioCloud server (https:// www. ezbio cloud. net/) 34 . Multiple sequences were aligned using the MEGA 7 software 35 , and CLUSTAL_W 36 . Using MEGA 7 software, phylogenetic trees were reconstructed according to the neighbor-joining (NJ) 37 , maximum-likelihood (ML) 38 , and maximum-parsimony (MP) 39 methods. Tree topology confidence levels were estimated by bootstrap analyses 40 based on 1,000 replications while evolutionary distances were calculated using Kimura's two-parameter model 41 . To obtain further taxonomic evidence, a UBCG phylogenomic tree based on the core gene set was reconstructed using the publicly available genomes of closely related taxa 42 . Based on 16S rRNA sequence similarity and phylogenetic analyses, a total of seven type strains of the genus Ramlibacter were selected for further chemotaxonomic tests and comparative analyses. R. monticola G-3-2 T , R. alkalitolerans CJ661 T , R. ginsenosidimutans BXN5-27 T , R. henchirensis TMB834 T , and R. tataouinensis TTB310 T were obtained from the KACC and R. humi 18 × 22-1 T and R. rhizophilus YS3.2.7 T were obtained from the KCTC.
Genomic DNA libraries of strains USB13 T , AW1 T , GTP1 T , and HM2 T were prepared using the TruSeq Nano DNA Library Prep kit (Illumina, USA) with an insert size of 350 bp. The draft genomes were sequenced by Macrogen Co., Ltd. (Seoul, South Korea) using the HiSeq X platform (Illumina, USA) and assembled using the SOAPdenovo version 3.10.1 de novo assembler 43 . DNA G + C content of the novel strains was calculated from genome data while genome contamination and completeness were assessed using the CheckM bioinformatics tool (https:// ecoge nomics. github. io/ CheckM). 44 For genome comparison, the known whole genome sequences of the Ramlibacter reference strains were obtained from the NCBI database while whole genomes of R. monticola KACC 19175 T , R. alkalitolerans KACC 19305 T , and R. ginsenosidimutans KACC 17527 T were sequenced and assembled using the same methods as those for the novel strains. Genome similarities of nucleotide sequences among the novel strains and their reference strains were determined by measuring the digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) values. ANI values were calculated using the OrthoANI calculator from EzBioCloud (https:// www. ezbio cloud. net/ tools/ ani) through pairwise comparisons between genome sequences based on the sequence analysis tool, USEARCH 45 , while dDDH values were obtained using the Genome-to-Genome Distance Calculator (GGDC; http:// ggdc. dsmz. de/ ggdc. php) based on calculations from Formula 2 46 , and AAI values were calculated using the online tool developed by the Kostas lab (http:// enve-omics. ce. gatech. edu/ aai/ index). The draft genome of USB13 T , AW1 T , GTP1 T , and HM2 T was annotated using the NCBI Prokaryote Genome Automatic Annotation Pipeline (PGAP), the Rapid Annotation using Subsystems Technology (RAST) server 47 , and the Cluster of Orthologous Groups of proteins (COG) database 48 . Identification of the CAZymes was done using the carbohydrate-active enzymes database (CAZy; http:// www. cazy. org/) 49 . The antiSMASH version 5.0 database was used to predict and identify existing secondary metabolism genes and biosynthetic gene clusters within the genome 50 . Additionally, a visual genomic comparison of the genomes of strains USB13 T , AW1 T , GTP1 T , and HM2 T was generated using the BLAST Ring Image Generator (BRIG) under default parameters (upper threshold, 70%; lower threshold, 50%; minimum threshold, 50%) with USB13 T as the reference genome 51 .
Microscopy of degraded filter paper. Samples were prepared to visualize morphological interactions between cellulose-degrading bacteria cells and cellulose fiber strands. Strains were first inoculated in 100 mL of basal salt medium containing Whatman Grade 1 qualitative filter paper (1 × 6 cm strip, 0.05 g per 20 mL) and incubated for 14 days at 30 °C. Following incubation, the degraded filter paper and bacterial cell mixture was loaded on round glass cover slips (Marienfeld, Germany) and fixed with 2.5% glutaraldehyde in 1X phosphate buffered saline (PBS; NaCl, 8 g/L; KCl, 0.2 g/L; Na 2 HPO 4 , 1.44 g/L; KH 2 PO 4 , 0.24 g/L; pH 7.4), according to the methods described by Ji et al. 52 . Subsequently, samples were washed three times with PBS buffer, followed by dehydration using a graded series of ethanol (30-100%). Samples were coated with platinum (15 nm; EM ACE200, Leica, Wetzlar, Germany) and examined under field emission-scanning electron microscopy (FE-SEM) analysis.
Crude enzyme production and enzymatic assay. Strains that showed positive results for CMC hydrolysis were cultured in basal salt medium containing Whatman Grade 1 qualitative filter paper (1 × 6 cm strip, 0.05 g per 20 mL) as the carbon source. Strains were incubated for a total of 7 days where cellulase activity was to be measured at 1, 3, 5, and 7 days following initial inoculation. On the days of testing, supernatant was collected and centrifuged at 5000 rpm for 15 min at 4 °C for enzyme activity analysis.
The activities of endoglucanase, exoglucanase, β-glucosidase, and total cellulase were determined by examining the amount of reducing sugar (glucose) released from CMC sodium salt, avicel, salicin, and filter paper, www.nature.com/scientificreports/ respectively. Enzyme activities were assessed in buffer solutions with different pH values (6.0, 7.0, 8.0) according to the dinitrosalicylic acid (DNS) method described by Ghose 53 . Endoglucanase, exoglucanase, and β-glucosidase activities were measured by incubating a mixture of 0.05 M citrate buffer, 0.5 mL of crude enzyme supernatant, and the corresponding substrates for 30 min at 50 °C. Total cellulase activity was measured by incubating 0.5 mL of crude enzyme supernatant with 1.0 mL of 0.05 M citrate buffer and one strip of Whatman Grade 1 qualitative filter paper (1 × 6 cm, approximately 50 mg) for 60 min at 50 °C. After incubation, the substrate hydrolysis reaction was terminated by adding 3 mL of DNS reagent. Results were measured spectrophotometrically where glucose standards were used to approximate the amount of glucose produced. All spectrophotometric data were measured using a UV-Vis spectrophotometer (U-3310, Hitachi, Tokyo, Japan). Enzyme activity was defined in international units (IU or U); one unit of enzymatic activity was defined as the amount of enzyme that releases 1 μmol of glucose per mL per 1 min of reaction. All experiments were conducted in triplicate and the data obtained were presented as mean values ± standard deviation.

Results and discussion
Chemotaxonomic characteristics. The predominant respiratory quinone for all novel strains was ubiquinone 8 (Q-8), consistent with other Ramlibacter species. C 16:0 and summed feature 3 (consisting of C 16:1 ω7c and/or C 16:1 ω6c) were identified as the common major fatty acids (> 10%) of the novel strains USB13 T , AW1 T , GTP1 T , and HM2 T . Other than the aforementioned fatty acids, strain USB13 T had C 10:0 3-OH additionally as its major fatty acid, whereas strains AW1 T and HM2 T shared C 17:0 cyclo and summed feature 8 (consisting of C 18:1 ω7c and/or C 18: 1 ω6c) as its additional fatty acids. Detailed comparisons of the fatty acid profiles of the novel strains and their reference strains are summarized in Table S1. Strains USB13 T , AW1 T , GTP1 T , and HM2 T shared major polar lipids diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), which was consistent with the major polar lipids of the reference strains. Additionally, the polar lipid profile of USB13 T consisted of one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids while the polar lipid profile of AW1 T had one unidentified lipid, one unidentified phosphoglycolipid, and three unidentified glycolipids in addition. The polar lipid profile of strain GTP1 T additionally consisted of two unidentified phosphoaminolipids, and that of strain HM2 T additionally had one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified phospholipids. Polar lipid profiles of the novel strains USB13 T , AW1 T , GTP1 T , and HM2 T are shown in Figure S1.
Physiological, morphological characteristics, and screening of cellulose-degrading strains. When grown on R2A agar, strain USB13 T produced reddish white and flat colonies while strain AW1 T produced orange, convex colonies, strain GTP1 T produced white, convex colonies, and strain HM2 T produced cream-colored, flat, transparent colonies. Under TEM, monotrichous flagella were observed only in strain HM2 T , and when tested for motility, strain USB13 T and AW1 T showed gliding motility, whereas strain GTP1 T was non-motile. Strains USB13 T and HM2 T showed positive results for both catalase and oxidase activities; strain AW1 T showed positive results for catalase and negative results for oxidase activity, and strain GTP1 T showed negative results for catalase and positive results for oxidase activity. All strains were identified to be strictly aerobic, while showing negative results for urea, gelatin, starch, chitin, and DNA hydrolysis and positive results for hydrolysis of Tween 80. In addition, strain USB13 T was the only strain to produce iron-chelating siderophores. When tested for NaCl tolerance, growth of strain USB13 T was observed in NaCl concentrations of 0-7% (w/v), possibly due to the fact the strain was isolated from a marine environment. A detailed comparison of physiological and morphological characteristics between the novel species and its closely related Ramlibacter strains is presented in Table 1, while TEM images of the novel strains are shown in Figure S2. Results of the reference strains in Table 1 coincided with the data from the original literature 1,3-5,7,8 .
R2A agar plates supplemented with 1% (w/v) CMC were stained with Congo red dye after 7 days of incubation. Clear zones only formed around colonies of strain USB13 T , indicating that strain USB13 T solely possessed CMC-hydrolyzing activity among the four novel strains. When inoculated in basal salt medium, filter paper from the USB13 T sample underwent degradation, whereas samples containing strains AW1 T , GTP1 T , and HM2 T did not show any signs of degradation.
Phylogenetic and genomic analyses. EzBioCloud search results and BLASTn searches revealed that the novel strains belonged to the family Comamonadaceae and genus Ramlibacter. Using BLASTn, 16S rRNA gene sequence similarities were determined where strain USB13 T was closest to strain GTP1 T (98.5%), followed by strain HM2 T (98.1%) and strain AW1 T (97.1%). Strain AW1 T shared the highest similarity with strain GTP1 T (97.3%), followed by strain HM2 T (97.1%), while strain GTP1 T shared a similarity of 98.2% with strain HM2 T . Phylogenetic analysis based on the MP method ( Fig. 1) showed the clustering of the novel strains USB13 T , AW1 T , GTP1 T , and HM2 T with strains such as R. monticola G-3-2 T , R. ginsenosidimutans BXN5-27 T , R. alkalitolerans CJ661 T , and R. rhizophilus YS3.2.7 T . Similar topologies were observed in trees reconstructed by ML (Figure S3) and MP methods. The UBCG phylogenomic tree (Fig. 2), which was reconstructed using whole genome sequences, also showed close clustering of the selected reference strains and novel strains. www.nature.com/scientificreports/ Draft genome sequences of the novel strains USB13 T , AW1 T , GTP1 T , and HM2 T were deposited in the Gen-Bank database under the accession numbers JACORT000000000, JAEQNA000000000, JACORU000000000, and JADDIV000000000, respectively. In addition, the draft genome sequences of R. monticola KACC 19175 T , R. alkalitolerans KACC 19305 T , and R. ginsenosidimutans KACC 17527 T were also deposited in GenBenk under the accession numbers JAEQNE000000000, JAEQND000000000, and JAEPWM000000000, respectively. The assembled genome size of the novel strains USB13 T , AW1 T , GTP1 T , and HM2 T was 5.53 Mbp, 5.11 Mbp, 6.15 Mbp, 4.31 Mbp, respectively. G + C content ranged from 67.9% to 69.9%, which was similar to those of the reference strains. The genomic features of the novel strains and their closely related Ramlibacter strains are presented in Table S2. CheckM analysis showed the following estimations for each strain: USB13 T , had a 99.84% completeness and 0.68% contamination; AWI T , had a 99.84% completeness and 0.86% contamination; GTP1 T , had a 99.38% Oxidase/catalase activity +/+ −/+ +/− +/+ −/+ +/+ +/+ +/+ +/+ +/+ +/+

Enzyme activities (API ZYM):
Assimilation of (20 NE):  54 . ANI values between the novel strains and their reference strains are presented in Fig. 3, while a detailed comparison of GGDC and AAI values are shown in Table 2.
Based on NCBI PGAP annotation and CAZyme prediction results, strain USB13 T , which was the only strain to show cellulolytic activity, possessed a total of four protein CDs encoding CAZymes, namely, two GH15 proteins, one glycosyl hydrolase protein, and one GH99-like domain-containing protein. Despite not showing any cellulolytic activity, strain AW1 T possessed eight CAZyme CDs; the most amount among the novel strains. The enzymes include, two GH2 proteins, one GH5 protein, three GH15 proteins, one glycoside hydrolase protein, and one cellulase family glycosyl hydrolase. Strain GTP1 T possessed two CDs encoding one GH15 protein and one GH16 protein; strain HM2 T possessed three CDs encoding one GH2, one GH15, and one GH18 protein.
COG predictions (Fig. 4) revealed that the majority of the core genes of the four novel strains accounted for genes belonging to the functional categories C (energy production and conversion), E (amino acid transport and metabolism), I (lipid transport and metabolism), T (signal transduction mechanisms), and K (transcription).  Table S5. AntiSMASH analysis results showed four gene clusters within the genome of strain USB13 T : ribosomally synthesized and post-translationally modified peptides (RIPP)-like cluster (989,516-1,000,916 nt; JACORT010000001), terpene synthesis (8,622-30,347 nt; JACORT010000003), RIPP precursor peptide recognition element (RRE)-containing cluster (311,469-333,619 nt; JACORT010000004), and redox-cofactor (281,860-303,948 nt; JACORT010000007). Among the clusters, the RRE-containing cluster showed 11% similarity to streptobactin, a tricatechol-type siderophore isolated from Streptomyces sp. YM5-799 56 293-130,892 nt), a signaling molecule known for its involvement in bacterial quorum sensing, the RIPP-like cluster (38,002-48,856 nt), and terpene synthesis (47,942-69,701 nt). Strain HM2 T had two gene clusters that encoded for resorcinol (403,967-445,901 nt), an organic compound known for its antiseptic properties, and terpene (697,660-721,242 nt), which showed 100% similarity for carotenoid synthesis. BRIG analysis results showed that a majority of the regions within the four analyzed genomes were conserved with at least 70% similarity ( Figure S4).

Cellulolytic potential and FE-SEM analysis of strain USB13 T . A USB13 T -inoculated basal salt
medium sample containing degraded filter paper was examined under FE-SEM to observe the morphological interactions between cellulose fibers and USB13 T cells. Images in Fig. 5 show individual rod cells of strain USB13 T surrounding filter paper fibers, indicating bacterial adherence.
The enzymatic assay results showed endoglucanase, exoglucanase, β-glucosidase, and filter paper cellulase (FPCase) activities of strain USB13 T , wherein activities for endoglucanase was the highest and β-glucosidase was the lowest in all experiments. As seen in Fig. 6A, enzyme activity for all cellulolytic enzymes increased along with its cultivation time. In addition, enzyme activities showed the highest results when tested on buffer solutions of pH 6.0 (Fig. 6B), indicating the enzymes' resistance to moderately acidic conditions. The pH of the buffer solution seemed to be an important factor in enzyme activity, as activity of endoglucanase, exoglucanase, and FPCase    www.nature.com/scientificreports/ Despite the absence of the main three cellulolytic enzymes, endoglucanase, exoglucanase, and β-glucosidase, the cellulolytic activity of strain USB13 T was confirmed through SEM images, CMC agar screening, and enzymatic assay results. However, because PGAP annotation results showed that other non-cellulolytic strains also possessed CAZymes, in some cases more than strain USB13 T , further research is necessary to understand the mechanics of how CAZymes and other cellulases interact to degrade cellulose, and how these genes are expressed under certain conditions. Furthermore, the cellulolytic activity of strain USB13 T can be further optimized for commercial use by adjusting growth conditions such as pH, temperature, and growth media.
While cellulolytic bacteria are known to inhabit animal intestinal tracts, the rumen, and soil, they can be found almost everywhere, such as ocean floors, municipal landfills, and even extreme environments such as hot springs 59 . In these habitats, cellulolytic bacteria utilize cellulose while cohabiting with non-cellulolytic bacteria. There have been many studies suggesting the synergistic role non-cellulolytic bacteria play in cellulose degradation, where non-cellulolytic bacteria aid cellulose degradation by neutralizing pH or removing harmful metabolites [60][61][62] .
Bacterial cellulases have shown immense value in various industries such as animal feed processing, food and brewery production, and agriculture, not to mention biofuel synthesis through biomass utilization 11 . Due to the versatile uses of bacterial cellulases, the cellulolytic strain USB13 T has the potential to become an invaluable  When observed on R2A agar, colonies are reddish white, flat with entire margins, and have a diameter of 1-2 mm. Growth of strain USB13 T is observed at 7-50 °C (optimum, 28-30 °C), at pH 5.0-10.0 (optimum, pH 6.0), and at NaCl concentrations of 0-7% (optimum, 0-3%). The strain is unable to grow in anaerobic conditions. Produces siderophores and hydrolyzes Tween 20, Tween 80, CMC, and esculin. According to the API ZYM results, the strain showed positive results for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, acid phosphatase, β-galactosidase, α-glucosidase, and β-glucosidase. In the API 20NE assay, strain USB13 T showed positive results only for β-galactosidase. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C 16:0 , C 10:0 3-OH, and summed feature 3 (consisting of C 16:1 ω7c and/or C 16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids. The G + C content is 69.7%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain USB13 T are MN603953 and JACORT000000000, respectively.
The type strain USB13 T (= KACC 21656 T = NBRC 114839 T ) was isolated from shallow coastal water at Haeundae Beach, Busan, Republic of Korea.
The type strain GTP1 T (= KACC 21702 T = NBRC 114488 T ) was isolated from soil at Seogwipo, Jeju Island, Republic of Korea.
Cells of strain HM2 T are Gram-negative, and positive for both oxidase and catalase activities. When observed on R2A agar, colonies are cream-colored, transparent, 1.0-2.5 mm in diameter, and flat with entire margins. Under TEM, monotrichous flagella are observed, and cells are rod-shaped with a width of 0.4-0.78 μm and length of 1.7-1.8 μm. The strain shows the fastest growth at a temperature range of 25-35 °C and at pH values between 8.0 and 9.0. When NaCl is present, growth is observed at concentrations of 0-3% (w/v), with optimal growth was observed at concentrations of 0-1% (w/v). The strain is not able to tolerate anaerobic conditions. Strain HM2 T hydrolyzes Tween 80 and weakly hydrolyzes casein. However, siderophore production cannot be observed when tested on CAS-blue agar. According to API ZYM tests, strain HM2 T shows positive results for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. In addition, API 20NE tests show positive results for nitrate (NO 3 ) to nitrite (NO 2 -) reduction and esculin hydrolysis. The G + C content is 69.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain HM2 T are MN498047 and JADDIV000000000, respectively.
The type strain HM2 T (= KCTC 82557 T = NBRC 114489 T ) was isolated from soil at Seopjikoji, Jeju Island, Republic of Korea.